Proteins and polypeptides are naturally occuring, and recently, synthetically created, compounds that are composed of long chains of amino acids. Proteins are found throughout living things and function as hormones, structural elements, enzymes, immunoglobulins, and other constituents of living things. Research regarding the structure and functions of proteins often requires that the amino acid sequence (primary structure) of the protein, be determined. In order for a protein, or parts of a protein such as somatostatin, insulin, endorphins, etc. to be synthesized chemically or by means of recombinant DNA techniques, the sequence of amino acids comprising such protein must usually be determined before a synthesis can be attempted. In a search involving the function of proteins, such as immunoglobulins, enzymes, viral coat proteins, and cell-surface proteins, the primary structure of the protein or polypeptide must be determined in an attempt to elucidate the mechanism of action of the protein. In recominant DNA methodology, the primary structure must be determined to elucidate a corresponding structure of a DNA or RNA encoding the same.
The primary sequence of amino acids in proteins or polypeptides is commonly determined by a stepwise chemical degradation process in which single amino acids are removed one by one from the end of the polypeptide for identification. The Edman degradation is the preferred method, while other methods have been developed and can be used in certain instances. In the Edman degradation, amino acid removal from the end of the protein is accomplished by reacting the N-terminal amino acid residue with a reagent which allows selective removal of that residue from the protein. The resulting amino acid derivative is converted into a stable compound which can be chemically removed from the reaction mixture and identified.
The stepwise degradation of peptides and proteins, as proposed by Edman in the 1950's (Edman, P., Acta Chem. Scand. 4,283 (1950)) has been altered little over the past 35 years. The three step process, as shown in FIG. 1, involves coupling the N-terminal amino acid of a peptide comprising N amino acids to phenylisothiocyanate (PITC) in a solvent under alkaline or anhydrous conditions (FIG. 1A). The excess reagent (a 500 to 10,000 fold molar excess) is removed by liquid-liquid extraction, (usually in multiple steps) and the solvent is removed in vacuo. The N-terminal amino acid is then subjected to cleavage by anhydrous acid to form an anilinothiazolinone (ATZ) derivative of the amino-terminal amino acid and the salt of a free peptide of amino acid of N-1 length which is the original peptide with the terminal amino acid removed. FIG. 1B. The cleavage acid is removed in vacuo and the ATZ derivative of the amino acid is extracted from the residual peptide which remains in the aqueous phase. The ATZ amino acid, which is highly unstable, is then subsequently subjected to conversion to a more stable phenylthiohydantoin (PTH) amino acid by reaction with aqueous acid. FIG. 1C. The shortened residual peptide is (N-1 amino acids) is then treated with PITC to initiate the next cycle of degradation. The resultant PTH amino acid then identified by chromatography. The foregoing steps are repeated for each terminal amino acid in the peptide.